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Stem Cells

Original Article
Mesenchymal Stem Cells in the Whartons Jelly
of the Human Umbilical Cord
Hwai-Shi Wang,a Shih-Chieh Hung,b,c Shu-Tine Peng,a Chun-Chieh Huang,a
Hung-Mu Wei,a Yi-Jhih Guo,a Yu-Show Fu,a Mei-Chun Lai,a Chin-Chang Chena
a

Institute of Anatomy and Cell Biology and bDepartment of Surgery, School of Medicine,
Yang-Ming University, Taipei, Taiwan, Republic of China; cDepartment of Orthopedics and Traumatology,
Veterans General Hospital-Taipei, Taipei, Taiwan, Republic of China
Key Words. Mesenchymal stem cells Whartons jelly Umbilical cord Multipotent cells

Abstract
The Whartons jelly of the umbilical cord contains
mucoid connective tissue and fibroblast-like cells. Using
flow cytometric analysis, we found that mesenchymal
cells isolated from the umbilical cord express matrix
receptors (CD44, CD105) and integrin markers (CD29,
CD51) but not hematopoietic lineage markers (CD34,
CD45). Interestingly, these cells also express significant
amounts of mesenchymal stem cell markers (SH2, SH3).
We therefore investigated the potential of these cells to
differentiate into cardiomyocytes by treating them with
5-azacytidine or by culturing them in cardiomyocyte-

conditioned medium and found that both sets of conditions resulted in the expression of cardiomyocyte markers, namely N-cadherin and cardiac troponin I. We also
showed that these cells have multilineage potential and
that, under suitable culture conditions, are able to differentiate into cells of the adipogenic and osteogenic lineages. These findings may have a significant impact on
studies of early human cardiac differentiation, functional
genomics, pharmacological testing, cell therapy, and tissue engineering by helping to eliminate worrying ethical
and technical issues. Stem Cells 2004;22:13301337

Introduction

maintains the tissue architecture of the umbilical cord by protecting it from pressure [4]. The phenotypic stromal cells in
the Whartons jelly are fibroblast-like cells [5]. However,
cells with the ultrastructural characteristics of myofibroblasts have been found [6]. Recently, Mitchell et al. [7] found
that matrix cells from Whartons jelly can be induced to form
neurons and glia cells by treating with basic fibroblast
growth factor and low-serum media plus butylated hydroxyanisole and dimethyl sulfoxide.
In this study, we demonstrate that mesenchymal cells
from the umbilical cord, when expanded in culture, express
adhesion molecules (CD44, CD105), integrin markers

The umbilical cord contains two arteries and one vein, which
are surrounded by mucoid connective tissue, and this is
called the Whartons jelly. The cord is covered by an epithelium derived from the enveloping amnion. The network of
glycoprotein microfibrils and collagen fibrils in the Whartons jelly has been previously studied [1]. The interlaced
collagen fibers and small, woven bundles are arranged to
form a continuous soft skeleton that encases the umbilical
vessels [2]. In the Whartons jelly, the most abundant glycosaminoglycan is hyaluronic acid [3], which forms a
hydrated gel around the fibroblasts and collagen fibrils and

Correspondence: Hwai-Shi Wang, Ph.D., Department of Anatomy,Yang-Ming University, 155, Sec. 2, Li-Nung Street, Taipei,
Taiwan, 112. Telephone: 886-2-28267035; Fax: 886-2-28283212; e-mail: hswang@ym.edu.tw Received January 27, 2004;
accepted for publication June 21, 2004. 1066-5099/2004/$12.00/0 doi: 10.1634/stemcells.2004-0013

STEM CELLS 2004;22:13301337

www.StemCells.com

Wang, Hung, Peng et al.

1331

(CD29, CD51), and mesenchymal stem cell (MSC) markers


(SH2, SH3) but not markers of hematopoietic differentiation
(CD34, CD45). After exposure of these cells to cardiomyocyte-conditioned medium or 5-azacytidine, they expressed
cardiac troponin-I and N-cadherin, indicating differentiation
into cardiomyocytes. Under suitable culture conditions,
these cells could also differentiate into osteogenic and adipogenic cells. Thus, human umbilical cord mesenchymal
cells can be expanded in culture and induced to form several
different types of cells. They may therefore prove to be a new
source of cells for cell therapy, including targets such as stromal tissue and cardiac muscle. This will help to avoid several
ethical and technical issues.

glucose (4.5 g/l) and 10% FBS for 57 days. Next, the cells
were trypsinized and suspended in DMEM at a concentration
of 5 106/ml, and then a 1-ml sample was incubated for 45
minutes at 4C with 150 l of various nonlabeled mouse antihuman antibodies followed by fluorescein isothiocyanate
(FITC)conjugated anti-mouse immunoglobulin G (IgG)
antibodies (10 mg/ml) for 1 hour at room temperature.
Finally, they were washed twice with phosphate-buffered
saline (PBS), pH 7.4; centrifuged; and fixed in 1.5 ml of 4%
paraformaldehyde. Control samples were incubated with
PBS instead of primary antibody. A FACScan machine (Becton, Dickinson, Franklin Lakes, NJ) was used to analyze antibody binding.

Materials and Methods

Induction of Adipogenic, Chondrogenic, or


Osteogenic Differentiation

Cell Culture
Institutional review board approval was obtained for all procedures. With the consent of the parents, fresh human umbilical cords were obtained after birth and stored in Hanks balanced salt solution for 124 hours before tissue processing to
obtain mesenchymal cells. After removal of blood vessels, the
mesenchymal tissue was scraped off from the Whartons jelly
with a scalpel and centrifuged at 250 g for 5 minutes at room
temperature and the pellet was washed with serum-free Dulbeccos modified Eagles medium (DMEM). Next, the cells
were centrifuged at 250 g for 5 minutes at room temperature
and then treated with collagenase (2 mg/ml) for 16 hours at
37C, washed, and treated with 2.5% trypsin for 30 minutes at
37C with agitation. Finally, the cells were washed and cultured in DMEM supplemented with 10% fetal bovine serum
(FBS) and glucose (4.5 g/l) in 5% CO2 in a 37C incubator.

Cultured cells at passage 2 were incubated in adipogenic differentiation medium (DMEM and 1 g/ml glucose [DMEMLG] containing 10% FBS, 50 g/ml of ascorbate-1 phosphate, 107 M dexamethasone, and 50 g/ml indomethacin
[Sigma, St. Louis]) in chondrogenic differentiation medium
(as cell pellets) (serum-free DMEM-LG containing insulintransferrin-selenium (ITS) + premix [GIBCO, Carlsbad, CA]
and 10 ng/ml transforming growth factor [TGF]-1 [Pepro
Tech, Rocky Hill, NJ]), in osteogenic differentiation medium
(DMEM-LG containing 10% FBS, 50 g/ml ascorbate-2
phosphate, 108 M dexamethasone, and 10 mM -glycerophosphate), or in DMEM-LG supplemented with 10% FBS as
a control. The medium was changed every 3 days, and the
cells, after completion of differentiation had been established
by morphology, were used for the histochemical staining and
immunohistochemistry studies.

Induction of Cardiogenic Differentiation


For 5-azacytidine treatment, the mesenchymal cells were
incubated for 24 hours in serum-free DMEM containing
3 M 5-azacytidine. The medium was then changed to
DMEM/10% FBS to prevent cell death due to prolonged
exposure to 5-azacytidine, and the cells were maintained in
DMEM/10% FBS for 3 days to 5 weeks. For treatment with
cardiomyocyte-conditioned medium, conditioned medium
was prepared by culturing cardiomyocytes, obtained from
the hearts of 7-day-old rats, in DMEM supplemented with
glucose (4.5 g/l) and 10% FBS for approximately 72 hours.
The cell culture medium was then harvested and centrifuged
at approximately 800 g for 10 minutes at room temperature,
and, finally, the supernatant was filtered for use as conditioned medium for the mesenchymal cells.

Flow Cytometry
After the mesenchymal cells were extracted from the umbilical cord, they were cultured in DMEM supplemented with

Immunocytochemistry
After the mesenchymal cells were extracted from the umbilical cord, they were cultured in DMEM supplemented with
glucose (4.5 g/l) and 10% FBS for 57 days, and then various
cell-differentiating factors were added for 3 days to 5 weeks.
Staining was performed on fixed, nonpermeabilized monolayers of mesenchymal cells grown on coverslips. The cells
were washed with PBS, fixed for 30 minutes at 37C in 0.05%
glutaraldehyde, and incubated for 25 minutes at room temperature with a 1:9 dilution of normal goat serum in PBS to block
nonspecific binding of the primary antibody. The slides were
then incubated for 75 minutes at 4C with various nonlabeled
mouse anti-human antibodies (10 g/ml) (Biosource International, Camarillo, CA) followed by FITC-coupled antimouse IgG antibodies (Vector, Burlingame, CA) for 1 hour at
room temperature. The coverslips were mounted with mounting medium (Vector) and viewed with a confocal laser-scanning microscope (Leica) using a 100/1.30 oil-immersion

Mesenchymal Stem Cells in Whartons Jelly

1332

objective lens and an appropriate filter. In the negative controls, in which the primary antibodies were omitted, negligible immunofluorescence was seen.

Western Blotting
For immunoblotting on polyvinylidene difluoride (PVDF)
membranes, cells from two 100-mm dishes per treatment
group were pooled, rinsed briefly with PBS, and then lysed at
room temperature for 10 minutes in 1 ml of PBS containing
1% sodium dodecyl sulfate, 0.5 mM phenylmethylsulfonyl
fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin, 5 g/ml pepstatin, 10 g/ml soybean trypsin inhibitor, and 0.5 mM dithiothreitol. After centrifugation for 20 minutes at 1,300 g and
4C, the supernatant was removed and centrifuged at 6,000 g
for 1 hour at 4C. The final supernatant was used as the cytosolic fraction, and the pellet was used as the membrane fraction. Equal amounts (50 mg of protein) of the membrane
fractions or cytosolic fractions were run on 12% polyacrylamide gels and transferred to PVDF membrane in transfer
buffer (four parts 25-mM Tris/200 mM buffer, pH 8.0, and
one part methanol). The membranes were then blocked for 2
hours at room temperature with 50 mM Tris HCl, 150 mM
NaCl, 0.05% Tween-20 (tris-buffered saline with Tween-20
[TBST]), pH 7.0, containing 5% nonfat dry milk and then
incubated overnight at 4C with rabbit anti-human cardiac troponin I monoclonal antibody (1:500) (Serotec, Oxford, U.K.)
or mouse anti-N-cadherin antibody (1:500) (Zymed, San
Francisco) in TBST. After three washes in TBST, the membranes were incubated with horseradish peroxidaseconjugated goat anti-rabbit IgG or goat anti-mouse IgG antibody at
a 1:1,000 dilution (Vector, Burlingame, CA), and bound antibody was detected by enhanced chemiluminescence (Amersham Life Sciences, Uppsala, Sweden).

Histochemical Staining
The medium was removed and the cells were washed twice
with PBS, fixed for 10 minutes at room temperature in 3.7%
paraformaldehyde, and washed twice with PBS. Cells
treated with the adipogenic and chondrogenic formulas were
stained with Oil red O or Toluidine blue to show adipogenic
or chondrogenic differentiation, respectively. Immunohistochemical staining for human type II collagen was also used to
demonstrate chondrogenic differentiation of the treated
cells. The mesenchymal cells treated by the osteogenic formula were stained with alkaline phosphatase staining and
von Kossa staining to reveal osteogenic differentiation.

RNA Extraction and Reverse Transcriptase


Polymerase Chain Reaction Analysis
Total RNA was extracted from untreated (control) and
treated cells using RNeasy Purification Reagent (Qiagen,

Valencia, CA), and then a sample (1 g) was reverse transcribed with Mmlv reverse transcriptase (RT) for 30 minutes
at 42C in the presence of oligo-dT primer. Polymerase chain
reaction (PCR) was performed using specific primers
designed from the published sequence of each cDNA as
follows: peroxisome proliferatoractivated receptor-2
(PPAR2) (595 bp), antisense 5'-CCTATTGACCCAGA
AAGCGATTC-3' and sense 5'-GCATTATGAGACATCCCCACTGC-3'; osteopontin (330 bp), antisense 5'-CAGTGA
CCAGTTCATCAGATTCATC-3' and sense 5'-CTAGGC
ATCACCTGTGCCATACC-3'; GAPDH (352 bp), antisense
5'-TCACGCCACAAGTTTCCCGGA-3' and sense 5'-CAC
CATCTTCCAGGAGCGAG-3'. PCR was performed for 30
to 35 cycles, with each cycle consisting of denaturation at
95C for 30 seconds, annealing at 55C to 63C for 30 seconds, and elongation at 72C for 1 minute, with an additional
10-minute incubation at 72C after completion of the last
cycle. To exclude the possibility of contaminating genomic
DNA, PCRs were also run without RT. The amplified cDNA
was separated by electrophoresis through a 1% agarose gel,
stained, and photographed under ultraviolet light.

Results
Characterization of Mesenchymal Cells
in Whartons Jelly
To determine whether stromal cells in the Whartons jelly of
the umbilical cord have multipotent potential, we extracted
cells from human umbilical cords and cultured them in
DMEM supplemented with glucose (4.5 mg/l) and 10%
FBS. The results reported are representative of the results
obtained using 30 umbilical cords. The cells demonstrated a
fibroblast-like phenotype (Fig. 1A). Flow cytometric analysis showed that the cells expressed high levels of matrix
markers (CD44, CD105), integrin markers (CD29, CD51),
and MSC markers (SH2, SH3) but did not express hematopoietic lineage markers (CD34, CD45) (Fig. 1B).

Cardiogenic Differentiation of Mesenchymal


Cells in Whartons Jelly
To investigate the potential of umbilical cord mesenchymal
cells to differentiate into cardiomyocytes, 5-azacytidine (3
mol/l), a drug that has been used to trigger this change [8],
was added to the culture medium. We stained the cells with
antibodies against human cardiac troponin I or F-actin and
examined the cells under confocal laser microscopy. As
shown in Figures 2A2D, cardiac troponin I was clearly more
strongly expressed in the differentiated cells than in untreated
cells. When umbilical cord mesenchymal cells were maintained in conditioned medium from primary rat cardiomyocyte cultures, the presence of N-cadherin expression was

Wang, Hung, Peng et al.

1333

Adipogenic Differentiation of Mesenchymal Cells


Adipogenic differentiation, as seen by Oil red Opositive
cells, was achieved by culturing the cells for 7 days in
DMEM-LG containing ascorbate, dexamethasome, and
indomethacin (Figs. 3B, 3C). When total RNA was isolated
and analyzed by RT-PCR, expression of NADPH, used as an
internal control, was the same in undifferentiated and differentiated cells, whereas the adipocyte marker PPAR2 was
not expressed in untreated cells but was expressed in adipogenic formula-treated cells (Fig. 3D).

Chondrogenic and Osteogenic Differentiation


of Mesenchymal Cells

Figure 1. Characteristics of mesenchymal cells from Whartons


jelly. (A): Cell cultures 3 days after the initial seeding of umbilical cord mesenchymal cells. The cells have a fibroblast-like morphology. (B): Flow cytometric analysis of surface-marker
expression on umbilical cord mesenchymal cells. The cells were
cultured in Dulbeccos modified Eagles medium supplemented
with 10% fetal bovine serum. After one passage at approximately
1 week, the cells were analyzed by flow cytometry. The shaded
area shows the profile of the negative control. The data shown are
representative of those obtained in six different experiments.

detected by staining at the cellcell junctions (Fig. 2F). Moreover, Western blots showed that newly extracted cells did not
express N-cadherin or cardiac troponin I but that both were
expressed in cells grown in conditioned medium. Cells grown
in DMEM with 10% FBS for 5 weeks expressed lower
amounts of N-cadherin and cardiac troponin I (Fig. 2G).

Chondrogenic differentiation of umbilical cord mesenchymal cells was achieved by pelleting the mesenchymal cells
and incubating them for 21 days with serum-free medium
supplemented with ITS + premix and 10 ng/ml of TGF-1.
The cell pellets developed chondrogenic characteristics after
treatment. Alcine blue staining of an aggrecan-rich extracellular matrix was evident in histological sections (Fig. 4A),
and a type II collagen-rich extracellular matrix was demonstrated immunohistochemically (Fig. 4B). However, the control groups were also positive for Alcine blue and type II collagen staining (data not shown). Osteogenic differentiation
of mesenchymal cells was seen as the formation of alkaline
phosphatasepositive aggregates and von Kossa stainpositive nodules (Fig. 4C), and this was achieved after 28 days of
culture in DMEM-LG containing 50 g/ml of ascorbate-2
phosphate, 108 M dexamethasone, and 10 mM -glycerophosphate. When total RNA was isolated and analyzed
by RT-PCR, expression of NADPH, used as an internal
control, was the same in undifferentiated and differentiated
cells, whereas osteopontin, an osteogenic marker, was not
expressed in newly isolated cells but was expressed in osteogenic formula-treated cells (Fig. 4D).

Discussion
MSCs were initially isolated from bone marrow by Friedenstein et al. [9] and then studied by other investigators [1016].
Recently, MSCs have been found to have the potential to differentiate into muscle cells, adipocytes, osteocytes [16], and
chondrocytes in culture [1618]. After systemic injection,
MSCs are incorporated into a variety of tissues, including
bone [19, 20], muscle [21], lung [19, 20, 22], and epithelium
[23]. Stem cells from bone marrow have also been shown to
form cardiomyocytes [2427]. The Whartons jelly of the
umbilical cord contains mucoid connective tissue and fibroblast-like cells. Using flow cytometric analysis, we found that
mesenchymal cells isolated from umbilical cord, when
expanded ex vivo, express matrix receptors (CD44, CD105)
and integrin markers (CD29, CD51) but not hematopoietic

1334

Mesenchymal Stem Cells in Whartons Jelly

Figure 2. Induction of cardiac troponin I and N-cadherin expression. (A, C): Expression of cardiac troponin-I (red) and F-actin filaments (green) in untreated mesenchymal cells. (B, D): Expression of cardiac troponin I and F-actin filaments in mesenchymal cells
incubated with serum-free DMEM containing 3 mol/l 5-azacytidine for 24 hours and DMEM/10% FBS for 21 days. (E): Immunofluorescence staining of N-cadherin in untreated umbilical cord mesenchymal cells. (F): N-cadherin expressed in the junctions between
mesenchymal cells (arrow) cultured in cardiomyocyte-conditioned medium for 5 weeks. A and B, bar = 40 m; C and D, bar = 10 m;
E and F, bar = 20 m. (G): Western blot showing N-cadherin and cardiac troponin I expression in, respectively, the membrane and
cytosolic fraction of untreated and conditioned medium-treated umbilical cord mesenchymal cells. Lane 1: newly extracted mesenchymal cells; lane 2: control cells cultured in DMEM supplemented with 10% FBS for 5 weeks; lane 3: cells grown in rat cardiomyocyteconditioned medium for 5 weeks. -actin was used as a loading control. Abbreviations: DMEM, Dulbeccos modified Eagles medium;
FBS, fetal bovine serum.

lineage markers (CD34, CD45). Interestingly, these cells also


express significant levels of some MSC markers (SH2, SH3)
(Fig. 1). These results suggest that stroma cells from Whartons jelly are similar to MSCs.
We investigated the potential of these cells to differentiate
into cardiomyocytes by treating them with 5-azacytidine or
maintaining them in cardiomyocyte-conditioned medium
and found that both treatments resulted in expression of the
cardiomyocyte markers N-cadherin and cardiac troponin I
(Fig. 2). Other cardiomyocyte-related markers, such as connexin 43, -actinin, and desmin, were also expressed after 5azacytidine treatment for 3 weeks (data not shown). Troponin
I, the inhibitory subunit of the troponin complex, is involved
in cardiac muscle contraction, whereas F-actin is an essential
cell cytoskeletal protein that maintains the structure of the
cardiac cell. Under confocal laser microscopy, cardiac troponin I was clearly more strongly expressed in the differentiated cells than in untreated cells (Fig. 2), suggesting that the
cells differentiated into cardiomyocytes rather than skeletal
cells, whereas F-actin expression in the differentiated cells
confirmed that the differentiated cells exhibited a specific
cell-attachment pattern similar to that of cardiomyocytes.
Undifferentiated cells can respond to signals from their
host tissue microenvironment and differentiate to produce

progeny with the phenotypic characteristics of the mature


cells of that tissue. N-cadherin is localized in the adherens
junction and is important for the assembly of the adherens
junction in cardiomyocytes [28, 29]. When umbilical cord
mesenchymal cells were maintained in conditioned medium
from primary rat cardiomyocyte cultures, Western blots
showed that the newly extracted cells did not express N-cadherin or cardiac troponin I but that both markers were
expressed in cells grown in conditioned medium and, to a
lesser extent, those grown in 10% FBS (Fig. 2E). This suggests that FBS provides some differentiating factors, but not
to the same extent as cardiomyocyte-conditioned medium.
However, it should be noted that there has been no report up to
the present that has described MSCs from adult bone marrow
and adipose tissue as able to spontaneously become cardiomyocytes in the control medium. It is possible that MSCs
from Whartons jelly are earlier-stage cells than MSCs from
adult bone marrow and adipose tissue. Thus, there may be a
fundamental difference between these cells from Whartons
jelly and the adult MSCs from bone marrow and adipose
tissue.
It has been found that after 5-azacytidine treatment for 2
weeks, murine bone marrow stromal cells can be differentiated into cardiomyocytes, which formed myotube-like struc-

Wang, Hung, Peng et al.

1335

Figure 3. Adipogenic differentiation of umbilical cord mesenchymal cells. (A): Control cells. (B): Cells in the adipogenic induction
group showing varying degrees of staining for Oil red O at 7 days of culture (bar = 10 m). (C): Control cells (bar = 25 m). (D): Cells in
the adipogenic induction group showing varying degrees of staining for Oil red O at 14 days of culture (bar = 25 m). (E): RT-PCR
analysis of untreated (lane 1) and adipogenic formula-treated (lane 2) cell cultures. Total RNA was analyzed by RT-PCR for PPAR2
(595 bp) and GAPDH (352 bp) mRNA. Abbreviations: PPAR2, peroxisome proliferatoractivated receptor-2; RT-PCR, reverse transcription polymerase chain reaction.

tures and began spontaneously beating [30]. However, in our


study, after 5-azacytidine treatment or culture in cardiomyocyte-conditioned media, the cells connected with adjoining
cells but did not form myotubes or start spontaneous contraction. The differentiation differences may possibly be
explained by the fact that the cells used in this study are
derived from a different species; namely, they are human
rather than mouse.
Under various culture conditions, these cells were able to
also differentiate into the chondrogenic, osteogenic, and adipogenic lineages. Adipogenic differentiation was seen as Oil
red Opositive cells and was also analyzed by RT-PCR, in
which the adipocyte marker PPAR2 was expressed in the
adipogenic formulatreated cells (Fig. 3). Chondrogenic differentiation of umbilical cord mesenchymal cells was
achieved by pelleting the mesenchymal cells and then incubating them with serum-free medium containing chondrogenic induction factors. The cell pellets expressed chondrogenic characteristics both in the control and after treatment
with chondrogenic induction formula. RT-PCR analysis of
chondrogenic markers Col2a1 and Col10a1 showed that
these were also expressed in the newly extracted mesenchymal cells (data not shown). Because umbilical cord is a supporting structure that contains an abundant amount of
hyaluronan, the mesenchymal cells from Whartons jelly

might have early chondrogenic characteristics in order to


give stiffness to the cord. Osteogenic differentiation of mesenchymal cells was seen as the formation of alkaline phosphatasepositive aggregates and mineral deposition staining
(Fig. 4). Osteopontin, an osteogenic marker, although not
expressed in newly isolated cells, was expressed in chondrogenic or osteogenic formulatreated cells (Fig. 4E). Taking
all these results together, the evidence suggests that multipotent cells exist in the umbilical cord.
The Whartons jelly contains colony-stimulating activity
[31] and high levels of insulin-like growth factor (IGF)-I and
IGF-1binding proteins [32], epidermal growth factor, TGF, and their common receptor genes [33]. Cultured human
MSCs spontaneously produce, or can be induced to produce,
cytokines for the support of hematopoietic cells [34, 35].
Thus, umbilical cord mesenchymal cells may have the characteristic ability of bone marrow MSCs to synthesize these
same cytokines for the support of hematopoietic cells. However, the possible existence of hematopoietic cells in the
umbilical cord needs to be investigated.
After exposure of these cells to 5-azacytidine or to cardiomyocyte-conditioned medium, the cells expressed both
cardiac troponin I and N-cadherin. These changes were
demonstrated by both immunofluorescent labeling and Western immunoblotting for cardiomyocyte markers. Because

1336

Mesenchymal Stem Cells in Whartons Jelly

Figure 4. Chondrogenic and osteogenic differentiation of umbilical cord mesenchymal cells. (A): Cells stained with Alcine blue after
chondrogenic induction for 21 days. Bar = 50 m. (B): Cells were treated as in (A) and then stained with antitype II collagen antibody.
Bar = 50 m. (C): Cells after osteogenic induction for 28 days showing varying degrees of positive staining for alkaline phosphatase
and von Kossa technique show the presence of mineral associated with the matrix. Bar = 8 m. (D): RT polymerase chain reaction
analysis of untreated cell cultures (lane 1), osteogenic formulatreated cell cultures (lane 2), and non-RT control (lane 3). Total RNA
was analyzed by osteopontin (330 bp) and GAPDH (352 bp) mRNA. Abbreviation: RT, reverse transcriptase.

umbilical cord mesenchymal cells have been used in cardiovascular tissue engineering [36], umbilical cord mesenchymal cells may be a good source for such use in cardiomyogenic differentiation. Our results also show that these cells
can be easily isolated and expanded in culture and induced to
differentiate, and this can result in the expression of phenotypes from a variety of lineages. These cells may therefore
prove to be a new source of cells for cellular therapies involving stromal tissue and, potentially, cardiac muscle repair. This
will avoid the ethical and technical issues involved in the use

of cells from other origins. These findings may have a significant impact on the study of cell therapy, early human cardiac
differentiation, functional genomics, pharmacological testing, and tissue engineering and potentially help to avoid worrying ethical issues.

Acknowledgments
We are grateful to Dr. S.L. Hsieh for providing us with antibodies for flow cytometry.

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